1. Volume 14, Issue 4, Pages 932-944 (February 2016)
Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels 
Go Kasuya, Yuichiro Fujiwara, Mizuki Takemoto, Naoshi Dohmae, Yoshiko Nakada-Nakura, Ryuichiro Ishitani, Motoyuki Hattori, Osamu Nureki 
Cell Reports 
Volume 14, Issue 4, Pages (February 2016)
DOI: /j.celrep
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2. Cell Reports 2016 14, 932-944DOI: (10.1016/j.celrep.2015.12.087)
Copyright © 2016 The Authors Terms and Conditions

3. Figure 1
Functional Properties of AmP2X and Crystallization Construct AmP2Xcryst
(A) FSEC profiles on a Superdex /300 GL column (GE Healthcare) for the EGFP-fused AmP2X WT (black), the EGFP-fused AmP2Xcryst (blue), and the EGFP-fused zfP2X4 WT (red), expressed in HEK293T cells. The arrows indicate the estimated elution positions of the void volume, the EGFP-fused P2X (trimer), and the free EGFP.
(B) Representative currents of the AmP2X WT evoked by 100 μM ATP.
(C) Representative current traces of AmP2Xcryst with the application of 100 μM ATP, showing no evoked current.
(D) Measurement of the ATP binding ability of the EGFP-fused AmP2Xcryst by a 3H-ATP binding assay. Error bars indicate ±SEM for triplicate samples. The calculated Kd for 3H-ATP binding is ± 0.238 μM.
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4. Figure 2 Overall Structure and ATP Binding Sites
(A) The ATP-bound AmP2Xcryst structure viewed parallel to the membrane. The omit Fo − Fc map contoured at 4σ, showing the electron density of the ATP.
(B and C) Close-up view of the ATP binding site in the ATP-bound AmP2Xcryst. The amino acid residues and ATP are depicted by stick models. The molecule is colored according to the dolphin-like model. Dotted black lines (C) indicate hydrogen bonds (<3.3 Å).
(D) Structural transition in the transmembrane helices. The transmembrane helices of the ATP-bound AmP2Xcryst (red) are superimposed on those of apo, closed ΔP2X4-B2 (gray) and of ATP-bound ΔP2X4-C (blue) using the Cα positions of residues 32–51 and 345–370 for AmP2X and residues 36–55 and 334–359 for zfP2X4, respectively. Gly350 in the two zfP2X4 structures and Val361 in the AmP2X structure are depicted by stick models.
(E and F) Structural comparison of the transmembrane domains as trimers viewed parallel to the membrane (E) and from the intracellular side (F). The transmembrane domain of the ATP-bound AmP2Xcryst (red) is superimposed as a trimer on those of apo, closed ΔP2X4-B2 (gray) and of ATP-bound ΔP2X4-C (blue) using the Cα positions of residues 32–51 and 345–370 for AmP2X and residues 36–55 and 334–359 for zfP2X4, respectively. The black arrows and bars denote the rotation of the transmembrane helices.
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5. Figure 3 Structural Comparisons of the Transmembrane Domains
(A–C) The transmembrane domains of apo, closed ΔP2X4-B2 (A); ATP-bound AmP2Xcryst (B); and ATP-bound ΔP2X4-C (C) viewed from the intracellular side. A surface model with a cartoon representation is shown for each structure. Amino acid residues involved in the pore constriction region are depicted by stick models. In (A) and (C), the distances between the Cα position of Ala347 and the center of the pore are shown by black lines and numbers (angstroms). In (B), the distances between the Cα position of Val361 and the center of the pore are shown by black lines and numbers (angstroms).
(D–F) Close-up views of the pore domain in apo, closed ΔP2X4-B2 (D); ATP-bound AmP2Xcryst (E); and ATP-bound ΔP2X4-C (F) viewed parallel to the membrane.
(G–I) Pore radii for apo, closed ΔP2X4-B2 (G); ATP-bound AmP2Xcryst (H); and ATP-bound ΔP2X4-C (I) along the pore center axis.
(J–L) Close-up views of the TM2 helices in apo, closed ΔP2X4-B2 (J); ATP-bound AmP2Xcryst (K); and ATP-bound ΔP2X4-C (L). Amino acid residues involved in the pore constriction region are depicted by stick models.
(M–O) Cartoon models of the transmembrane regions in apo, closed ΔP2X4-B2 (M); ATP-bound AmP2Xcryst (N); and ATP-bound ΔP2X4-C (O).
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6. Figure 4 Metal Binding Sites of AmP2X and of apo, Closed zfP2X4
(A) Overall view of the zinc binding sites in the AmP2X structure. The omit Fo − Fc map contoured at 6σ, showing the electron densities of the zinc ions.
(B–D) Close-up views of the zinc binding sites. The anomalous difference Fourier maps contoured at 4σ, showing the electron densities of the zinc ions.
(E) Overall view of the gadolinium binding site in the apo, closed zfP2X4 structure (PDB: 3H9V).
(F and G) Close-up view of the gadolinium binding site. The superscripts “zf,” “am,” and “h3” refer to zfP2X4, AmP2X, and human P2X3, respectively. The amino acid residues zfGlu98 and zfAsp99 correspond to amGlu106 and amSer107 (h3Thr87), respectively.
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7. Figure 5 Zn2+ and Mg2+ Effects on the M1 and M2 Sites
(A) Representative currents of rat P2X4 (rWT) and the zinc binding site mutants evoked by 3 μM ATP in the presence and absence of 10 μM Zn2+.
(B) Accumulated data for the relative current amplitudes in the presence and absence of Zn2+. Bars depict mean + SEM (n = 5–7). The superscripts “r” and “am” refer to the rat P2X4 and AmP2X, respectively. The amino acid residues rGlu95 and rAsp170 correspond to amGlu106 and amAsp188, respectively.
(C–F) Effects of MgCl2 on the ATP-activated currents in human P2X1 WT (hWT) and the hD170A/hD171A (amD188A/amY189A) mutant. (C and D) Representative currents of hWT under the 5 mM MgCl2 conditions (C) and under the 0.5 mM MgCl2 conditions (D). (E and F) Representative currents of hD170A/hD171A (amD188A/amY189A) mutant under the 5 mM MgCl2 conditions (E) and under the 0.5 mM MgCl2 conditions (F). The numbers above the currents indicate the ATP concentrations.
(G) Concentration-response relationships of ATP-evoked currents of hWT under the 5 mM MgCl2 conditions (EC50 = 1.65 ± 0.40 μM, n = 6) and under the 0.5 mM MgCl2 conditions (EC50 = 1.14 ± 0.09 μM, n = 6) and of the hD170A/hD171A (amD188A/amY189A) mutant under the 5 mM MgCl2 conditions (EC50 = 6.55 ± 1.44 μM, n = 7) and under the 0.5 mM MgCl2 conditions (EC50 = 1.34 ± 0.27 μM, n = 6). Error bars depict mean ± SEM. The superscripts “h” and “am” refer to the human P2X1 and AmP2X, respectively. The amino acid residues hAsp170 and hAsp171 correspond to amAsp188 and amTyr189, respectively.
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8. Figure 6 Zinc Modulation Mechanism
(A–C) Distributions of r (A), θ (B), and ϕ (C) are displayed. Orange distributions indicate the results of the MD simulations in the presence of Zn2+ at the M1 site, and cyan distributions indicate the results of the MD simulations in the absence of Zn2+ at the M1 site.
(D and E) The transmembrane domain of ATP-bound AmP2Xcryst after the 150 ns MD simulations in the presence of Zn2+ at the M1 site (D) and in the absence of Zn2+ at the M1 site (E). Each structure is viewed from the extracellular side. A surface model containing the cartoon representation is shown for each structure.
(F) The time course of the pore sizes of ATP-bound AmP2Xcryst through the 150 ns MD simulations. The orange graph shows the results of the simulations in the presence of Zn2+ at the M1 site, and the cyan graph shows the results of the simulations in the absence of Zn2+ at the M1 site. The pore sizes are defined by the triangular dimensions formed by the three Cα positions of Val362 from each subunit.
(G–J) Subunit comparisons of ATP-bound AmP2Xcryst before the MD simulation (gray) and after the 150 ns MD simulations in the presence of Zn2+ at the M1 sites (orange) and in the absence of Zn2+ at the M1 sites (cyan). Close-up views of the M1 site (H), the lower body (I), and the fluke (J) are shown in each box.
(K–M) Cartoon models of the Zn2+-dependent stabilization mechanism of the ATP-bound AmP2Xcryst open state. (K) The model after the 150 ns MD simulation in the absence of Zn2+ at the M1 site. (L) The crystal structure model of ATP, Zn2+-bound AmP2Xcryst. (M) The model after the 150 ns MD simulation in the presence of Zn2+ at the M1 site. In cartoon models (K) and (M), the black arrows denote the movement from the crystal structure model (L).
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